Tryptophanyl-tRNA synthetase-derived polypeptides useful for the regulation of angiogenesis

ABSTRACT

The invention provides a method for inhibiting ocular neovascularization in a patient. The method comprises administering to a patient an ocular neovascularization inhibiting amount of a water-soluble polypeptide selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 7, and an ocular neovascularization inhibiting fragment thereof, which includes at least one of amino acid residue signature sequences HVGH (SEQ ID NO:10) and KMSAS (SEQ ID NO:11). A method for assaying the angiogenesis inhibiting activity of a composition is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.10/982,014, filed Nov. 4, 2004, now U.S. Pat. No. 7,273,844, which is acontinuation-in-part of U.S. patent application Ser. No. 10/240,527,filed Sep. 30, 2002, now U.S. Pat. No. 7,144,984, which is the NationalStage of PCT/US01/08966, filed Mar. 21, 2001, which claims the benefitof U.S. Provisional Application for Patent Ser. No. 60/193,471 filedMar. 31, 2000; and a continuation-in-part of U.S. patent applicationSer. No. 10/240,532, filed Sep. 30, 2002, now U.S. Pat. No. 7,067,126,which is the National Stage of PCT/US01/08975, filed Mar. 21, 2001,which also claims the benefit of U.S. Provisional Application for PatentSer. No. 60/193,471 filed Mar. 31, 2000; and a continuation-in-part ofU.S. patent application Ser. No. 10/080,839, filed Feb. 22, 2002, nowabandoned, which claims the benefit of U.S. Provisional Application forPatent Ser. No. 60/270,951 filed Feb. 23, 2001; the disclosures of whichare incorporated herein by reference.

GOVERNMENTAL RIGHTS

This invention was made with governmental support from the United StatesGovernment, National Institutes of Health, Grant GM23562. The UnitedStates Government has certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to compositions comprising truncated tRNAsynthetase polypeptides, as well as nucleic acids encoding suchtruncated tRNA synthetase polypeptides. Methods of making and using suchcompositions are also disclosed.

BACKGROUND OF THE INVENTION

Aminoacyl-tRNA synthetases, which catalyze the aminoacylation of tRNAmolecules, are ancient proteins that are essential for decoding geneticinformation during the process of translation. In higher eukaryotes,nine aminoacyl-tRNA synthetases associate with at least three otherpolypeptides to form a supramolecular multienzyme complex (Mirande etal., Eur. J. Biochem. 147:281-89 (1985)). Each of the eukaryotic tRNAsynthetases consists of a core enzyme, which is closely related to theprokaryotic counterpart of the tRNA synthetase, and an additional domainthat is appended to the amino-terminal or carboxyl-terminal end of thecore enzyme (Mirande, Prog. Nucleic Acid Res. Mol. Biol. 40:95-142(1991)).

In most cases, the appended domains appear to contribute to the assemblyof the multienzyme complex. However, the presence of an extra domain isnot strictly correlated with the association of a synthetase into themultienzyme complex.

Mammalian TrpRS molecules have an amino-terminal appended domain. Innormal human cells, two forms of TrpRS can be detected: a major formconsisting of the full-length molecule (amino acid residues 1-471 of SEQID NO: 1) and a minor truncated form (“mini TrpRS”; amino acid residues1-424 of SEQ ID NO: 3). The minor form is generated by the deletion ofthe amino-terminal domain through alternative splicing of the pre-mRNA(Tolstrup et al., J. Biol. Chem. 270:397-403 (1995)). The amino-terminusof mini TrpRS has been determined to be the methionine residue atposition 48 of the full-length TrpRS molecule (i.e., residue 48 of SEQID NO: 1). Alternatively, truncated TrpRS can be generated byproteolysis (Lemaire et al., Eur. J. Biochem. 51:237-52 (1975)). Forexample, bovine TrpRS is highly expressed in the pancreas and issecreted into the pancreatic juice (Kisselev, Biochimie 75:1027-39(1993)), thus resulting in the production of a truncated TrpRS molecule.These results suggest that truncated TrpRS can have a function otherthan the aminoacylation of tRNA (id.)

Angiogenesis, or the proliferation of new capillaries from pre-existingblood vessels, is a fundamental process necessary for embryonicdevelopment, subsequent growth, and tissue repair. Angiogenesis is aprerequisite for the development and differentiation of the vasculartree, as well as for a wide variety of fundamental physiologicalprocesses including embryogenesis, somatic growth, tissue and organrepair and regeneration, cyclical growth of the corpus luteum andendometrium, and development and differentiation of the nervous system.In the female reproductive system, angiogenesis occurs in the follicleduring its development, in the corpus luteum following ovulation and inthe placenta to establish and maintain pregnancy. Angiogenesisadditionally occurs as part of the body's repair processes, e.g. in thehealing of wounds and fractures. Angiogenesis is also a factor in tumorgrowth, since a tumor must continuously stimulate growth of newcapillary blood vessels in order to grow. Angiogenesis is an essentialpart of the growth of human solid cancer, and abnormal angiogenesis isassociated with other diseases such as rheumatoid arthritis, psoriasis,and diabetic retinopathy (Folkman, J. and Klagsbrun, M., Science235:442-447 (1987)).

Several factors are involved in angiogenesis. Both acidic and basicfibroblast growth factor molecules that are mitogens for endothelialcells and other cell types. Angiotropin and angiogenin can induceangiogenesis, although their functions are unclear (Folkman, J., CancerMedicine, pp. 153-170, Lea and Febiger Press (1993)). A highly selectivemitogen for vascular endothelial cells is vascular endothelial growthfactor or VEGF (Ferrara, N., et al., Endocr. Rev. 13:19-32, (1992)).

The vast majority of diseases that cause catastrophic loss of vision doso as a result of ocular neovascularization; age related maculardegeneration (ARMD) affects 12-15 million American over the age of 65and causes visual loss in 10-15% of them as a direct effect of choroidal(sub-retinal) neovascularization. The leading cause of visual loss forAmericans under the age of 65 is diabetes; 16 million individuals in theUnited States are diabetic and 40,000 per year suffer from ocularcomplications of the disease, often a result of retinalneovascularization. While laser photocoagulation has been effective inpreventing severe visual loss in subgroups of high risk diabeticpatients, the overall 10-year incidence of retinopathy remainssubstantially unchanged. For patients with choroidal neovascularizationdue to ARMD or inflammatory eye disease such as ocular histoplasmosis,photocoagulation, with few exceptions, is ineffective in preventingvisual loss. While recently developed, non-destructive photodynamictherapies hold promise for temporarily reducing individual loss inpatients with previously untreatable choroidal neovascularization, only61.4% of patients treated every 3-4 months had improved or stabilizedvision compared to 45.9% of the placebo-treated group.

In the normal adult, angiogenesis is tightly regulated, and is limitedto wound healing, pregnancy and uterine cycling. Angiogenesis is turnedon by specific angiogenic molecules such as basic and acidic fibroblastgrowth factor (FGF), vascular endothelial growth factor (VEGF),angiogenin, transforming growth factor (TGF), tumor necrosis factor-α(TNF-α) and platelet derived growth factor (PDGF). Angiogenesis can besuppressed by inhibitory molecules such as interferon-α,thrombospondin-1, angiostatin and endostatin. It is the balance of thesenaturally occurring stimulators and inhibitors that controls thenormally quiescent capillary vasculature. When this balance is upset, asin certain disease states, capillary endothelial cells are induced toproliferate, migrate and ultimately differentiate.

Angiogenesis plays a central role in a variety of disease includingcancer and ocular neovascularization. Sustained growth and metastasis ofa variety of tumors has also been shown to be dependent on the growth ofnew host blood vessels into the tumor in response to tumor derivedangiogenic factors. Proliferation of new blood vessels in response to avariety of stimuli occurs as the dominant finding in the majority of eyedisease and that blind including proliferative diabetic retinopathy(PDR), ARMD, rubeotic glaucoma, interstitial keratitis and retinopathyof prematurity. In these diseases, tissue damage can stimulate releaseof angiogenic factors resulting in capillary proliferation. VEGF plays adominant role in iris neovascularization and neovascular retinopathies.While reports clearly show a correlation between intraocular VEGF levelsand ischemic retinopathic ocular neovascularization, FGF likely plays arole. Basic and acidic FGF are known to be present in the normal adultretina, even though detectable levels are not consistently correlatedwith neovascularization. This can be largely due to the fact that FGFbinds very tightly to charged components of the extracellular matrix andcannot be readily available in a freely diffusible form that would bedetected by standard assays of intraocular fluids.

A final common pathway in the angiogenic response involvesintegrin-mediated information exchange between a proliferating vascularendothelial cell and the extracellular matrix. This class of adhesionreceptors, called integrins, are expressed as heterodimers having an αand β subunit on all cells. One such integrin, α_(v)β₃, is the mostpromiscuous member of this family and allows endothelial cells tointeract with a wide variety of extracellular matrix components. Peptideand antibody antagonists of this integrin inhibit angiogenesis byselectively inducing apoptosis of the proliferating vascular endothelialcells. Two cytokine-dependent pathways of angiogenesis exist and can bedefined by their dependency on distinct vascular cell integrins, α_(v)β₃and α_(v)β₅. Specifically, basic FGF- and VEGF-induced angiogenesisdepend on integrin α_(v)β₃ and α_(v)β₅, respectively, since antibodyantagonists of each integrin selectively block one of these angiogenicpathways in the rabbit corneal and chick chorioallantoic membrane (CAM)models. Peptide antagonists that block all α_(v) integrins inhibit FGF-and VEGF-stimulated angiogenesis. While normal human ocular bloodvessels do not display either integrin, α_(v)β₃ and α_(v)β₅ integrinsare selectively displayed on blood vessels in tissues from patients withactive neovascular eye disease. While only α_(v)β₃ was consistentlyobserved in tissue from patients with ARMD, α_(v)β₃ and α_(v)β₅ bothwere present in tissues from patients with PDR. Systemicallyadministered peptide antagonists of integrins blocked new blood vesselformation in a mouse model of retinal vasculogenesis.

Hence, anti-angiogenic agents have a role in treating retinaldegeneration to prevent the damaging effects of these trophic and growthfactors. Angiogenic agents also have a role in promoting desirablevascularization to retard retinal degeneration by enhancing blood flowto cells.

SUMMARY OF THE INVENTION

Tryptophanyl-tRNA synthetase-derived polypeptides, shorter than the onesthat occur in nature, have chemokine activity and are useful forresearch, diagnostic, prognostic and therapeutic applications. In oneembodiment, these tRNA synthetase-derived polypeptides are useful forregulating vascular endothelial cell function, and in particular, forinhibiting angiogenesis, especially ocular neovascularization. Thepolypeptide has the amino acid residue sequence shown in SEQ ID NO: 7,SEQ ID NO: 12, or an angiogenesis inhibiting fragment thereof, thefragment including at least one of amino acid residue signaturesequences HVGH (SEQ ID NO: 10) and KMSAS (SEQ ID NO: 11).

These truncated tryptophanyl-tRNA synthetase (TrpRS)-derivedpolypeptides have an amino-terminal truncation, but can include aRossmann fold nucleotide binding domain. These polypeptides are capableof regulating vascular endothelial cell function.

A preferred truncated TrpRS-derived polypeptide is a fragment of humanTrpRS consisting of the amino acid residue sequence of SEQ ID NO: 12(i.e., amino acid residues 94-471 of SEQ ID NO: 1). Another preferredTrpRS-derived polypeptide consists of the amino acid residue sequence ofSEQ ID NO: 7 (i.e., the His₆-tagged version of SEQ ID NO: 12). Preferredangiogenesis-inhibiting fragments of SEQ ID NO: 12 or SEQ ID NO: 7include fragments bracketed by the signature sequences shown in SEQ IDNO: 10 and SEQ ID NO: 11, or that include at least one of thesesignature sequences.

In another embodiment, the invention comprises an isolatedpolynucleotide consisting of a nucleotide sequence at least 95%identical to the sequence of a polynucleotide selected from the groupconsisting of a polynucleotide of SEQ ID NO: 6, a polynucleotide whichis hybridizable to a polynucleotide of SEQ ID NO: 6; a polynucleotideencoding the polypeptide of SEQ ID NO: 7; a polynucleotide encoding thepolypeptide of SEQ ID NO: 12, a polynucleotide encoding a polypeptideepitope of SEQ ID NO: 7; and a polynucleotide that is hybridizable to apolynucleotide encoding a polypeptide epitope of SEQ ID NO: 7. Thepresent invention also includes a recombinant expression vectorcomprising an isolated nucleic acid molecule that encodes any of theaforementioned tryptophanyl-tRNA synthetase-derived polypeptides.Another embodiment is a host cell comprising such a recombinantexpression vector.

The invention additionally provides composition and dosage forms thatinclude the truncated tryptophanyl-tRNA synthetase-derived polypeptidestogether with a pharmaceutically suitable excipient. Such compositionsare suitable for intraocular, e.g., intravitreal, sub-retinal or thelike, as well as for systemic administration, e.g., transdermal,transmucosal, enteral or parenteral administration.

In another embodiment, the invention provides a method of treatingneovascular eye diseases such as age-related macular degeneration,ocular complications of diabetes, rubeotic glaucoma, retinopathy ofprematurity, keratitis, ischemic, retinopathy (e.g., sickle cell),pathological myopic, ocular histoplasmosis, pterygia, punitate innerchoroidopathy, and the like, by administering an angiogenesis inhibitingamount of the polypeptide together with an appropriate, physiologicallycompatible excipient or carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid residue sequence of tryptophanyl-t-RNAsynthetase polypeptide (SEQ ID NO: 1) with included signature sequences(SEQ ID NO: 10 & SEQ ID NO: 11), shown in a box, encompassed also withinthe truncated form (amino acid residue sequences 94-471 of SEQ ID NO:1).

FIG. 2 is a photomicrograph that illustrates the ability of T2 toinhibit vascularization of the secondary deep network of the mouseretina. Row A shows the vascular network of a retina exposed to fulllength TrpRS. Row B shows the vascular network of a retina exposed toMini-TrpRS. Row C shows the vascular network of a retina exposed topolypeptide T2 of the present invention. The first (left) column of eachRow shows the primary superficial network, and the second column showsthe secondary deep network.

FIG. 3 is a graphical representation of data reported in Example 3,below.

FIG. 4 is a graphical representation of data reported in Example 4,below.

FIG. 5 shows a photomicrographs illustrating the binding localization ofa fragment of TrpRS (T2) in the retina in a mouse model. Thedistribution of the injected protein was restricted to blood vessels asconfirmed by co-staining labeled T2 treated eyes with anfluorescein-labeled (ALEXA® 594) anti-collagen IV antibody. Panel Ashows a retinal cross-section five days after injection offluorescein-labeled T2 (on Day P12), the green fluorescence of thelabeled T2 was still visible, indicated by the arrows; only a primaryvascular layer (1°) was observed. Panel B shows that retinas injected onP7 with fluorescein-labeled full-length TrpRS developed a secondaryvascular layer (2°) in addition to the primary layer (1°) by P12 but novascular staining was observed. Panel C shows cross-sectioned slices ofnormal neonatal retinas stained with fluorescein-labeled T2;fluorescein-labeled T2 bound only to blood vessels within the primaryand secondary vascular layers (indicated by the arrows). Panel D showsthat no retinal vessel staining was observed when fluorescein-labeledfull-length TrpRS was applied to the retinas in either the primary orthe secondary vascular layers.

FIG. 6 shows the nucleic acid sequence encoding for the T1 fragment ofhuman TrpRS, SEQ ID NO: 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

“Truncated tRNA synthetase polypeptides” means polypeptides that areshorter than the corresponding full length tRNA synthetase.

“TrpRS” means tryptophanyl-tRNA synthetase.

“Cell culture” encompasses both the culture medium and the culturedcells.

The phrase “isolating a polypeptide from the cell culture” encompassesisolating a soluble or secreted polypeptide from the culture medium aswell as isolating an integral membrane protein from the cultured cells.

“Cell extract” includes culture media, especially spent culture mediafrom which the cells have been removed. A cell extract that contains theDNA or protein of interest should be understood to mean a homogenatepreparation or cell-free preparation obtained from cells that expressthe protein or contain the DNA of interest.

“Plasmid” is an autonomous, self-replicating extrachromosomal DNAmolecule and is designated by a lower case “p” preceded and/or followedby capital letters and/or numbers. The starting plasmids herein areeither commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but can vary in accordance with thesupplier's instructions. After digestion the reaction is subjected toelectrophoresis directly on a poly-acrylamide gel to isolate the desiredfragment. The nucleotides present in various DNA and RNA fragments aredesignated herein by the standard single letter designations (A, T, C,G, U) used in the art.

“Polynucleotide” embodying the present invention can be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA can be double-stranded or single-stranded, and ifsingle stranded can be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide can beidentical to the coding sequence shown in SEQ ID NO: 6 or can be adifferent coding sequence which coding sequence, as a result of theredundancy or degeneracy of the genetic code, encodes the same, maturepolypeptide sequence shown in SEQ ID NO: 7.

The term “Polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

“Oligonucleotides” refers to either a single stranded polynucleotide ortwo complementary polynucleotide strands which can be chemicallysynthesized. Such synthetic oligonucleotides have no 5′ phosphate andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Amino acid residue” refers to an amino acid which is part of apolypeptide. The amino acid residues described herein are preferably inthe L″ isomeric form. However, residues in the D″ isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property is retained by the polypeptide. NH₂ refers to thefree amino group present at the amino terminus of a polypeptide. COOHrefers to the free carboxyl group present at the carboxyl terminus of apolypeptide. I

All amino acid residue sequences represented herein by formulae have aleft to right orientation in the conventional direction ofamino-terminus to carboxyl-terminus. In addition, the phrase “amino acidresidue” is broadly defined to include the 20 amino acids commonly foundin natural proteins, as well as modified and unusual amino acids, suchas those referred to in 37 C.F.R. § § 1.821-1.822, and incorporatedherein by reference. A dash at the beginning or end of an amino acidresidue sequence indicates a peptide bond to a further sequence of oneor more amino acid residues or to an amino-terminal group such as NH₂ orto a carboxyl-terminal group such as COOH.

In a peptide or protein, suitable conservative substitutions of aminoacids are known to those of skill in this art and can be made generallywithout altering the biological activity of the resulting molecule.Those of skill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. Co., p. 224).

Such substitutions are preferably made in accordance with those setforth in TABLE 1 as follows:

TABLE 1 Original residue Conservative substitution Ala (A) Gly; Ser Arg(R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G)Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T)Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; LeuOther substitutions are also permissible and can be determinedempirically or in accord with known conservative substitutions.

“Complementing plasmid” describes plasmid vectors that deliver nucleicacids into a packaging cell line for stable integration into achromosome in the cellular genome.

“Delivery plasmid” is a plasmid vector that carries or delivers nucleicacids encoding a therapeutic gene or gene that encodes a therapeuticproduct or a precursor thereof or a regulatory gene or other factor thatresults in a therapeutic effect when delivered in vivo in or into a cellline, such as, but not limited to a packaging cell line, to propagatetherapeutic viral vectors.

A variety of vectors is described herein. For example, one vector isused to deliver particular nucleic acid molecules into a packaging cellline for stable integration into a chromosome. These types of vectorsare generally identified herein as complementing plasmids. A furthertype of vector described herein carries or delivers nucleic acidmolecules in or into a cell line (e.g., a packaging cell line) for thepurpose of propagating therapeutic viral vectors; hence, these vectorsare generally referred to herein as delivery plasmids. A third “type” ofvector described herein is used to carry nucleic acid molecules encodingtherapeutic proteins or polypeptides or regulatory proteins or areregulatory sequences to specific cells or cell types in a subject inneed of treatment; these vectors are generally identified herein astherapeutic viral vectors or recombinant adenoviral vectors or viralAd-derived vectors and are in the form of a virus particle encapsulatinga viral nucleic acid containing an expression cassette for expressingthe therapeutic gene.

“DNA or nucleic acid homolog” refers to a nucleic acid that includes apreselected conserved nucleotide sequence, such as a sequence encoding atherapeutic polypeptide. The term “substantially homologous” refers to apolypeptide having at least 80%, preferably at least 90%, mostpreferably at least 95% homology therewith or a less percentage ofhomology or identity and conserved biological activity or function.

The terms “homology” and “identity” are often used interchangeably. Inthis regard; degree of homology or identity can be determined, forexample, by comparing sequence information using a GAP computer program.The GAP program utilizes the alignment method of Needleman and Wunsch,J. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman, Adv.Appl. Math. 2:482 (1981). Briefly, the GAP program defines similarity asthe number of aligned symbols (i.e., nucleotides or amino acids) whichare similar, divided by the total number of symbols in the shorter ofthe two sequences. The preferred default parameters for the GAP programcan include: (1) a unary comparison matrix (containing a value of 1 foridentities and 0 for non-identities) and the weighted comparison matrixof Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1986), as describedby Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358 (1979); (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps. Whether any twonucleic acid molecules have nucleotide sequences that are at least 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determined usingknown computer algorithms such as the “FAST A” program, using forexample, the default parameters as in Pearson and Lipman, Proc. Natl.Acad. Sci. USA 85:2444 (1988). Alternatively the BLAST function of theNational Center for Biotechnology Information database can be used todetermine identity. In general, sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. (See, e.g.:Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, (1988); Smith, D. W., ed., Biocomputing: Informaticsand Genome Projects, Academic Press, New York, (1993); Griffin, A. M.,and Griffin, H. G., eds., Computer Analysis of Sequence Data, Part I,Humana Press, New Jersey, (1994); von Heinje, G., Sequence Analysis inMolecular Biology, Academic Press, (1987); and Gribskov, M. andDevereux, J., eds., Sequence Analysis Primer, M Stockton Press, NewYork, (1991)). While there exist a number of methods to measure identitybetween two polynucleotide or polypeptide sequences, the term “identity”is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J.Applied Math. 48:1073 (1988)). Methods commonly employed to determineidentity or similarity between two sequences include, but are notlimited to, those disclosed in Martin J. Bishop, ed., Guide to HugeComputers, Academic Press, San Diego, (1994), and Carillo, H. & Lipton,D., SIAM J. Applied Math. 48:1073 (1988). Methods to determine identityand similarity are codified in computer programs. Preferred computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, GCG program package(Devereux, J., et al., Nucleic Acids Research 12 (I):387 (1984)),BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J. Molec. Biol. 215:403(1990)).

The term “identity” represents a comparison between a test and areference polypeptide or polynucleotide. For example, a test polypeptidecan be defined as any polypeptide that is 90% or more identical to areference polypeptide. As used herein, the term at least “90% identicalto” refers to percent identities from 90 to 99.99 relative to thereference polypeptides. Identity at a level of 90% or more is indicativeof the fact that, assuming for exemplification purposes a test andreference polynucleotide length of 100 amino acids are compared. No morethan 10% (i.e., 10 out of 100) amino acids in the test polypeptidediffers from that of the reference polypeptides. Similar comparisons canbe made between a test and reference polynucleotides. Such differencescan be represented as point mutations randomly distributed over theentire length of an amino acid sequence or they can be clustered in oneor more locations of varying length up to the maximum allowable, e.g.10/100 amino acid difference (approximately 90% identity). Differencesare defined as nucleic acid or amino acid substitutions, or deletions.

The terms “gene therapy” and “genetic therapy” refer to the transfer ofheterologous DNA to the certain cells, target cells, of a mammal,particularly a human, with a disorder or conditions for which suchtherapy is sought. The DNA is introduced into the selected target cellsin a manner such that the heterologous DNA is expressed and atherapeutic product encoded thereby is produced. Alternatively, theheterologous DNA can in some manner mediate expression of DNA thatencodes the therapeutic product, it can encode a product, such as apeptide or RNA that in some manner mediates, directly or indirectly,expression of a therapeutic product. Genetic therapy can also be used tonucleic acid encoding a gene product replace a defective gene orsupplement a gene product produced by the mammal or the cell in which itis introduced. The introduced nucleic acid can encode a therapeuticcompound, such as a growth factor inhibitor thereof, or a tumor necrosisfactor or inhibitor thereof, such as a receptor therefor, that is notnormally produced in the mammalian host or that is not produced intherapeutically effective amounts or at a therapeutically useful time.The heterologous DNA encoding the therapeutic product can be modifiedprior to introduction into the cells of the afflicted host in order toenhance or otherwise alter the product or expression thereof.

“Heterologous DNA” is DNA that encodes RNA and proteins that are notnormally produced in vivo by the cell in which it is expressed or thatmediates or encodes mediators that alter expression of endogenous DNA byaffecting transcription, translation, or other regulatable biochemicalprocesses. Heterologous DNA can also be referred to as foreign DNA. AnyDNA that one of skill in the art would recognize or consider asheterologous or foreign to the cell in which it is expressed is hereinencompassed by the term “heterologous DNA”. Examples of heterologous DNAinclude, but are not limited to, DNA that encodes traceable markerproteins, such as a protein that confers drug resistance, DNA thatencodes therapeutically effective substances, such as anti-canceragents, enzymes and hormones, and DNA that encodes other types ofproteins, such as antibodies. Antibodies that are encoded byheterologous DNA can be secreted or expressed on the surface of the cellin which the heterologous DNA has been introduced. Hence, “heterologousDNA” or “foreign DNA”, refers to a DNA molecule not present in the exactorientation and position as the counterpart DNA molecule found in thecorresponding wild-type adenovirus. It can also refer to a DNA moleculefrom another organism or species (i.e., exogenous) or from anotheradenovirus (Ad) serotype.

“Therapeutically effective DNA product” is a product that is encoded byheterologous DNA so that, upon introduction of the DNA into a host, aproduct is expressed that effectively ameliorates or eliminates thesymptoms, manifestations of an inherited or acquired disease, or thatcures said disease. Typically, DNA encoding the desired heterologous DNAis cloned into a plasmid vector and introduced by routine methods, suchas calcium-phosphate mediated DNA uptake or microinjection, intoproducer cells, such as packaging cells. After amplification in producercells, the vectors that contain the heterologous DNA are introduced intoselected target cells.

“Expression or delivery vector” refers to any plasmid or virus intowhich a foreign or heterologous DNA can be inserted for expression in asuitable host cell, i.e., the protein or polypeptide encoded by the DNAis synthesized in the host cell's system. Vectors capable of directingthe expression of DNA segments (genes) encoding one or more proteins arereferred to herein as “expression vectors.” Also included are vectorsthat allow cloning of cDNA (complementary DNA) from mRNAs produced usingreverse transcriptase.

“Gene” is a nucleic acid molecule whose nucleotide sequence encodes RNAor polypeptide. A gene can be either RNA or DNA. Genes can includeregions preceding and following the coding region (leader and trailer)as well as intervening sequences (introns) between individual codingsegments (exons).

“Isolated” with reference to a nucleic acid molecule, polypeptide, orother biomolecule, means that the nucleic acid or polypeptide hasseparated from the genetic environment from which the polypeptide ornucleic acid were obtained. It can also mean altered from the naturalstate. For example, a polynucleotide or a polypeptide naturally presentin a living animal is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. Thus, a polypeptide orpolynucleotide produced and/or contained within a recombinant host cellis considered isolated. Also intended as an “isolated polypeptide” or an“isolated polynucleotide” are polypeptides or polynucleotides that havebeen purified, partially or substantially, from a recombinant host cellor from a native source. For example, a recombinantly produced versionof a compounds can be substantially purified by the one-step methoddescribed in Smith and Johnson, Gene 67:31-40 (1988). The terms“isolated” and “purified” are sometimes used interchangeably. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

By “isolated polynucleotide” is meant that the nucleic acid is free ofthe coding sequences of those genes that, in the naturally-occurringgenome of the organism (if any) immediately flank the gene encoding thenucleic acid of interest. Isolated DNA can be single-stranded ordouble-stranded, and can be genomic DNA, cDNA, recombinant hybrid DNA,or synthetic DNA. It can be identical to a native DNA sequence, or candiffer from such sequence by the deletion, addition, or substitution ofone or more nucleotides.

“Isolated” or “purified” as it refers to preparations made frombiological cells or hosts means any cell extract containing theindicated DNA or protein including a crude extract of the DNA or proteinof interest. For example, in the case of a protein, a purifiedpreparation can be obtained following an individual technique or aseries of preparative or biochemical techniques and the DNA or proteinof interest can be present at various degrees of purity in thesepreparations. The procedures can include for example, but are notlimited to, ammonium sulfate fractionation, gel filtration, ion exchangechange chromatography, affinity chromatography, density gradientcentrifugation and electrophoresis.

A preparation of DNA or protein that is “substantially pure” or“isolated” means a preparation free from naturally occurring materialswith which such DNA or protein is normally associated in nature.“Essentially pure” should be understood to mean a “highly” purifiedpreparation that contains at least 95% of the DNA or protein ofinterest.

“Packaging cell line” is a cell line that provides a missing geneproduct or its equivalent.

“Adenovirus viral particle” is the minimal structural or functional unitof a virus. A virus can refer to a single particle, a stock of particlesor a viral genome. The adenovirus (Ad) particle is relatively complexand can be resolved into various substructures.

“Post-transcription regulatory element (PRE)” is a regulatory elementfound in viral or cellular messenger RNA that is not spliced, i.e.intronless messages. Examples include, but are not limited to, humanhepatitis virus, woodchuck hepatitis virus, the TK gene and mousehistone gene. The PRE can be placed before a polyA sequence and after aheterologous DNA sequence.

“Pseudotyping” describes the production of adenoviral vectors havingmodified capsid protein or capsid proteins from a different serotypethan the serotype of the vector itself. One example, is the productionof an adenovirus 5 vector particle containing an Ad37 fiber protein.This can be accomplished by producing the adenoviral vector in packagingcell lines expressing different fiber proteins.

“Promoters of interest herein” can be inducible or constitutive.Inducible promoters will initiate transcription only in the presence ofan additional molecule; constitutive promoters do not require thepresence of any additional molecule to regulate gene expression. aregulatable or inducible promoter can also be described as a promoterwhere the rate or extent of RNA polymerase binding and initiation ismodulated by external stimuli. Such stimuli include, but are not limitedto various compounds or compositions, light, heat, stress and chemicalenergy sources. Inducible, suppressible and repressible promoters areconsidered regulatable promoters. Preferred promoters herein, arepromoters that are selectively expressed in ocular cells, particularlyphotoreceptor cells.

“Receptor” refers to a biologically active molecule that specificallybinds to (or with) other molecules. The term “receptor protein” can beused to more specifically indicate the proteinaceous nature of aspecific receptor.

“Recombinant” refers to any progeny formed as the result of geneticengineering. This can also be used to describe a virus formed byrecombination of plasmids in a packaging cell.

“Transgene” or “therapeutic nucleic acid molecule” includes DNA and RNAmolecules encoding an RNA or polypeptide. Such molecules can be “native”or naturally-derived sequences; they can also be “non-native” or“foreign” that are naturally- or recombinantly-derived. The term“transgene,” which can be used interchangeably herein with the term“therapeutic nucleic acid molecule,” is often used to describe aheterologous or foreign (exogenous) gene that is carried by a viralvector and transduced into a host cell. Therapeutic nucleotide nucleicacid molecules include antisense sequences or nucleotide sequences whichcan be transcribed into antisense sequences. Therapeutic nucleotidesequences (or transgenes) all include nucleic acids that function toproduce a desired effect in the cell or cell nucleus into which saidtherapeutic sequences are delivered. For example, a therapeutic nucleicacid molecule can include a sequence of nucleotides that encodes afunctional protein intended for delivery into a cell which is unable toproduce that functional protein.

“Vitreous of the eye” refers to a material that fills the chamber behindthe lens of the eye (i.e., vitreous humor or vitreous body).

“Promoter region” refers to the portion of DNA of a gene that controlstranscription of the DNA to which it is operatively linked. The promoterregion includes specific sequences of DNA that are sufficient for RNApolymerase recognition, binding and transcription initiation. Thisportion of the promoter region is referred to as the promoter. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of the RNApolymerase. These sequences can be cis acting or can be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, can be constitutive or regulated.

“Operatively linked” means that the sequences or segments have beencovalently joined into one piece of DNA, whether in single or doublestranded form, whereby control sequences on one segment controlexpression or replication or other such control of other segments. Thetwo segments are not necessarily contiguous, however.

“Package” refers to a solid matrix or material such as glass, plastic(e.g., polyethylene, polypropylene or polycarbonate), paper, foil andthe like capable of holding within fixed limits a polypeptide,polyclonal antibody, or monoclonal antibody of the present invention.Thus, for example, a package can be a glass vial used to containmilligram quantities of a contemplated polypeptide or it can be amicrotiter plate well to which microgram quantities of a contemplatedpolypeptide or antibody have been operatively affixed (i.e., linked) soas to be capable of being immunologically bound by an antibody orantigen, respectively.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

“Diagnostic system” in the context of the present invention alsoincludes a label or indicating means capable of signaling the formationof an immunocomplex containing a polypeptide or antibody molecule of thepresent invention.

“Complex” as used herein refers to the product of a specific bindingreaction such as an antibody-antigen or receptor-ligand reaction.Exemplary complexes are immunoreaction products.

“Label” and “Indicating means” in their various grammatical forms referto single atoms and molecules that are either directly or indirectlyinvolved in the production of a detectable signal to indicate thepresence of a complex. Any label or indicating means can be linked to orincorporated in an expressed protein, polypeptide, or antibody moleculethat is part of an antibody or monoclonal antibody composition of thepresent invention or used separately, and those atoms or molecules canbe used alone or in conjunction with additional reagents. Such labelsare themselves well-known in clinical diagnostic chemistry andconstitute a part of this invention only insofar as they are utilizedwith otherwise novel proteins methods and/or systems.

Discussion

The polypeptide shown in FIG. 1 as amino acid residues 94-471 of SEQ IDNO: 1 (e.g., SEQ ID NO: 12), as well as that having the amino acidsequence of SEQ ID NO: 7, or the polypeptide encoded by the cDNA of SEQID NO: 6, constitute parts of the present invention. In addition tovariants of the above polypeptides, the present invention also includesvariants of polynucleotides. Included polynucleotide variants can be anaturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide. Thus, the presentinvention includes polynucleotides encoding the same polypeptide asshown in SEQ ID NO: 7, SEQ ID NO: 12, and the polypeptide encoded by thecDNA of SEQ ID NO: 6 as well as variants of such polynucleotides whichvariants encode for an angiogenesis inhibiting fragment, derivative oranalog of the polypeptides of SEQ ID NO: 7 and SEQ ID NO: 12. Suchnucleotide variants include deletion variants, substitution variants andaddition or insertion variants.

As indicated above, the polynucleotide can have a coding sequence whichis a naturally occurring allelic variant of the coding sequence shown inSEQ ID NO: 6. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

As used herein, the term T1 refers to both the polypeptide of SEQ ID NO:13 and to the His₆-tagged polypeptide of SEQ ID NO: 5. A cDNA encodingthe polypeptide of SEQ ID NO 5 is SEQ ID NO: 4 (FIG. 6). The term T2, asuses herein, refers to both the polypeptide of SEQ ID NO: 12 and to theHis₆-tagged polypeptide of SEQ ID NO: 7. The term TrpRS, as uses herein,refers to both the polypeptide consisting of amino acid residues 1-471of SEQ ID NO: 1 and to the His₆-tagged polypeptide of SEQ ID NO: 1.

The present invention also includes polynucleotides wherein the codingsequence for the mature polypeptide can be fused in the same readingframe to a polynucleotide which aids in expression and secretion of apolypeptide from a host cell, for example, a leader sequence whichfunctions as a secretory sequence for controlling transport of apolypeptide from the cell. The polypeptide having a leader sequence is apreprotein and can have the leader sequence cleaved by the host cell toform the mature form of the polypeptide. The polynucleotides can alsoencode for a proprotein which is the mature protein plus additional 5′amino acid residues. A mature protein having a prosequence is aproprotein and is an inactive form of the protein. Once the prosequenceis cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention canencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and presequence (leadersequence).

The polynucleotides of the present invention can also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence can be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencecan be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably 70% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term “stringent conditions” means hybridization will occur only ifthere is at least 95% and preferably at least 97% identity between thesequences. The polynucleotides which hybridize to the hereinabovedescribed polynucleotides in a preferred embodiment encode polypeptideswhich retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNA of SEQ ID NO: 6.

When referring to the polypeptide of SEQ ID NO: 7, SEQ ID NO: 12, or toa polypeptide encoded by the polynucleotide of SEQ ID NO: 6, the terms“fragment,” “derivative” and “analog” mean a polypeptide portion whichretains substantially the same angiostatic (i.e., angiogenesisinhibiting) function or activity as such polypeptide. Thus, an “analog”includes a proprotein which can be activated by cleavage of theproprotein portion to produce an angiostatically active maturepolypeptide.

The polypeptide of the present invention can be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The angiogenesis inhibiting fragment, derivative or analog of thepolypeptide of SEQ ID NO: 7, SEQ ID NO: 12, or the polypeptide encodedby the polynucleotide of SEQ ID NO: 6 can be (i) one in which one ormore of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue can or can not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe polypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe polypeptide, such as a leader or secretory sequence or a sequencewhich is employed for purification of the polypeptide or a proproteinsequence. Such fragments, derivatives and analogs are deemed to bewithin the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The present invention also includes vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which can be, forexample, a cloning vector or an expression vector. The vector can be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the tRNA synthetase polypeptide genes. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention can be utilized forproducing corresponding polypeptides by recombinant techniques. Thus,for example, the polynucleotide sequence can be included in any one of avariety of expression vehicles, in particular vectors or plasmids forexpressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; yeast plasmids; vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, and pseudorabies. A preferred vector ispET20b. However, any other plasmid or vector can be used as long as itis replicable and viable in the host.

As described above, the appropriate DNA sequence can be inserted intothe vector by a variety of procedures. In general, the DNA sequence isinserted into an appropriate restriction endonuclease sites byprocedures known in the art. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. Representative examples of such promoters include LTR or SV40promoter, the E. coli lac or trp promoters, the phage lambda P_(L)promoter and other promoters known to control expression of genes inprokaryotic or eukaryotic cells or their viruses. The expression vectoralso contains a ribosome binding site for translation initiation and atranscription terminator. The vector can also include appropriatesequences for amplifying expression.

In addition, the expression vectors preferably contain a gene to providea phenotypic trait for selection of transformed host cells such asdihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as herein abovedescribed, as well as an appropriate promoter or control sequence, canbe employed to transform an appropriate host to permit the host toexpress the protein. Representative examples of appropriate hostsinclude bacterial cells, such as E. coli, Salmonella typhimurium,Streptomyces; fungal cells, such as yeast; insect cells, such asDrosophila and Sf9; animal cells such as CHO, COS or Bowes melanoma;plant cells, etc. The selection of an appropriate host is deemed to bewithin the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example: Bacterial: pQE70, pQE-9 (Qiagen), pBs,phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a,pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, PRIT5(Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, PMSG, pSVL (Pharmacia) and pET20B. In one preferredembodiment, the vector is pET20B. However, any other plasmid or vectorcan be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), PLand trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described construct. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, 1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Proteins can be expressed in mammalian cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell-free translationsystems can also be employed to produce such proteins using RNAs derivedfrom the DNA constructs of the present invention. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook. et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure ofwhich is hereby incorporated by reference.

Transcription of a DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to about 300 base pairs (bp), that act on apromoter to increase its transcription. Examples include the SV40enhancer on the late side of the replication origin (bp 100 to 270), acytomegalovirus early promoter enhancer, a polyoma enhancer on the lateside of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 viralgenome, for example, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites can be used to provide the required nontranscribedgenetic elements.

Polypeptides are recovered and purified from recombinant cell culturesby methods used heretofore, including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxyapatite chromatography and lectinchromatography. It is preferred to have low concentrations(approximately 0.1-5 mM) of calcium ion present during purification(Price, et al., J. Biol. Chem., 244:917 (1969)). Protein refolding stepscan be used, as necessary, in completing configuration of the matureprotein. Finally, high performance liquid chromatography (HPLC) can beemployed for final purification steps.

The polypeptides of the present invention can be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention can beglycosylated with mammalian or other eukaryotic carbohydrates or can benon-glycosylated.

The polypeptides of the present invention can be modified to improvestability and increase potency by means known in the art. For example,L-amino acids can be replaced by D-amino acids, the amino terminus canbe acetylated, or the carboxyl terminus modified, e.g.,ethylamine-capped (Dawson, D. W., et al., Mol. Pharmacol., 55: 332-338(1999)).

The polypeptide of the present invention can also be employed as genetherapy in accordance with the present invention by expression of suchpolypeptide in vivo.

Various viral vectors that can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, adeno-associatedvirus (AAV), or, preferably, an RNA virus such as a retrovirus.Preferably, the retroviral vector is a derivative of a murine or avianretrovirus, or is a lentiviral vector. The preferred retroviral vectoris a lentiviral vector. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. By inserting a zinc finger derived-DNA binding polypeptidesequence of interest into the viral vector, along with another gene thatencodes the ligand for a receptor on a specific target cell, forexample, the vector is made target specific. Retroviral vectors can bemade target specific by inserting, for example, a polynucleotideencoding a protein. Preferred targeting is accomplished by using anantibody to target the retroviral vector. Those of skill in the art willknow of, or can readily ascertain without undue experimentation,specific polynucleotide sequences which can be inserted into theretroviral genome to allow target specific delivery of the retroviralvector containing the zinc finger-nucleotide binding proteinpolynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsitation. Helper cell lines which havedeletions of the packaging signal include but are not limited to Ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced. The vector virions produced by thismethod can then be used to infect a tissue cell line, such as NIH 3T3cells, to produce large quantities of chimeric retroviral virions.

Another targeted delivery system for polynucleotides encoding zincfinger derived-DNA binding polypeptides is a colloidal dispersionsystem. Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Thepreferred colloidal system of this invention is a liposome. Liposomesare artificial membrane vesicles which are useful as delivery vehiclesin vitro and in vivo. It has been shown that large unilamellar vesicles(LUV), which range in size from 0.2-4.0 μm can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand be delivered to cells in a biologically active form (Fraley, et al.,Trends Biochem. Sci., 6:77, (1981)). In addition to mammalian cells,liposomes have been used for delivery of polynucleotides in plant, yeastand bacterial cells. In order for a liposome to be an efficient genetransfer vehicle, the following characteristics should be present: (1)encapsulation of the genes of interest at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, (1988)).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids can also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes has been classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system can be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

In general, the compounds bound to the surface of the targeted deliverysystem will be ligands and receptors which will allow the targeteddelivery system to find and “home in” on the desired cells. A ligand canbe any compound of interest which will bind to another compound, such asa receptor.

In general, surface membrane proteins which bind to specific effectormolecules are referred to as receptors. In the present invention,antibodies are preferred receptors. Antibodies can be used to targetliposomes to specific cell-surface ligands. For example, certainantigens expressed specifically on tumor cells, referred to astumor-associated antigens (TAAs), can be exploited for the purpose oftargeting antibody-zinc finger-nucleotide binding protein-containingliposomes directly to the malignant tumor. Since the zincfinger-nucleotide binding protein gene product can be indiscriminatewith respect to cell type in its action, a targeted delivery systemoffers a significant improvement over randomly injecting non-specificliposomes. A number of procedures can be used to covalently attacheither polyclonal or monoclonal antibodies to a liposome bilayer.Antibody-targeted liposomes can include monoclonal or polyclonalantibodies or fragments thereof such as Fab, or F(ab′)₂, as long as theybind efficiently to an the antigenic epitope on the target cells.Liposomes can also be targeted to cells expressing receptors forhormones or other serum factors.

There are available to one skilled in the art multiple viral andnon-viral methods suitable for introduction of a nucleic acid moleculeinto a target cell. Genetic manipulation of primary tumor cells is wellknown in the art. Genetic modification of a cell can be accomplishedusing one or more techniques well known in the gene therapy field(Mulligan, R. C. Human Gene Therapy, 5(4):543-563 (1993)). Viraltransduction methods can comprise the use of a recombinant DNA or an RNAvirus comprising a nucleic acid sequence that drives or inhibitsexpression of a protein having sialyltransferase activity to infect atarget cell. A suitable DNA virus for use in the present inventionincludes but is not limited to an adenovirus (Ad), adeno-associatedvirus (AAV), herpes virus, vaccinia virus or a polio virus. A suitableRNA virus for use in the present invention includes but is not limitedto a retrovirus or Sindbis virus. It is to be understood by thoseskilled in the art that several such DNA and RNA viruses exist that canbe suitable for use in the present invention.

Adenoviral vectors are useful for gene transfer into eukaryotic cells,to study eukaryotic gene expression, for vaccine development, and inanimal models. Ad-mediated gene therapy has also been utilized inhumans, such as for the transfer of the cystic fibrosis transmembraneconductance regulator (CFTR) gene to the lung. Routes for administratingrecombinant Ad to different tissues in vivo include, for example,intratracheal instillation, injection into muscle, peripheralintravenous injection and stereotactic inoculation to brain. Theadenoviral vector, then, is widely available to one skilled in the artand is suitable for use in the present invention.

Adeno-associated virus (AAV) has recently been introduced as a genetransfer system with potential applications in gene therapy. Wild-typeAAV has been reported to demonstrate high-level infectivity, broad hostrange and specificity in integrating into the host cell genome. Herpessimplex virus type-1 (HSV-1) is attractive as a vector system,especially for use in the nervous system because of its neurotropicproperty. Vaccinia virus, of the poxvirus family, has also beendeveloped as an expression vector. Each of the above-described vectorsare widely available to one skilled in the art and would be suitable foruse in the present invention.

Retroviral vectors are capable of infecting a large percentage of thetarget cells and integrating into the cell genome. Retroviruses weredeveloped as gene transfer vectors relatively earlier than otherviruses, and were first used successfully for gene marking andtransducing the cDNA of adenosine deaminase (ADA) into humanlymphocytes. Preferred retroviruses include lentiviruses. In preferredembodiments, the retrovirus is selected from the group consisting ofHIV, BIV and SIV.

“Non-viral” delivery techniques that have been used or proposed for genetherapy include DNA-ligand complexes, adenovirus-ligand-DNA complexes,direct injection of DNA, CaPO₄ precipitation, gene gun techniques,electroporation, liposomes and lipofection. Any of these methods arewidely available to one skilled in the art and would be suitable for usein the present invention. Other suitable methods are available to oneskilled in the art, and it is to be understood that the presentinvention can be accomplished using any of the available methods oftransfection. Several such methodologies have been utilized by thoseskilled in the art with varying success. Lipofection can be accomplishedby encapsulating an isolated DNA molecule within a liposomal particleand contacting the liposomal particle with the cell membrane of thetarget cell. Liposomes are self-assembling, colloidal particles in whicha lipid bilayer, composed of amphiphilic molecules such as phosphatidylserine or phosphatidyl choline, encapsulates a portion of thesurrounding media such that the lipid bilayer surrounds a hydrophilicinterior. Unilammellar or multilammellar liposomes can be constructedsuch that the interior contains a desired chemical, drug, or, as in theinstant invention, an isolated DNA molecule.

The cells can be transfected in vivo, ex vivo, or in vitro. The cellscan be transfected as primary cells isolated from a patient or a cellline derived from primary cells, and are not necessarily autologous tothe patient to whom the cells are ultimately administered. Following exvivo or in vitro transfection, the cells can be implanted into a host.

In order to obtain transcription of the nucleic acid of the presentinvention within a target cell, a transcriptional regulatory regioncapable of driving gene expression in the target cell is utilized. Thetranscriptional regulatory region can comprise a promoter, enhancer,silencer or repressor element and is functionally associated with anucleic acid of the present invention. Preferably, the transcriptionalregulatory region drives high level gene expression in the target cell.Transcriptional regulatory regions suitable for use in the presentinvention include but are not limited to the human cytomegalovirus (CMV)immediate-early enhancer/promoter, the SV40 early enhancer/promoter, theJC polyomavirus promoter, the albumin promoter, PGK and the α-actinpromoter coupled to the CMV enhancer.

The vectors of the present invention can be constructed using standardrecombinant techniques widely available to one skilled in the art. Suchtechniques can be found in common molecular biology references such asSambrook, et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press (1989), D. Goeddel, ed., Gene ExpressionTechnology, Methods in Enzymology series, Vol. 185, Academic Press, SanDiego, Calif. (1991), and Innis, et al. PCR Protocols. A Guide toMethods and Applications Academic Press, San Diego, Calif. (1990).

Administration of a polypeptide or a nucleic acid of the presentinvention to a target cell in vivo can be accomplished using any of avariety of techniques well known to those skilled in the art.

The vectors and compositions of the present invention can beadministered orally, parentally, by inhalation spray, rectally, ortopically in dosage unit formulations containing conventionalpharmaceutically acceptable carriers, adjuvants, and vehicles. The termparenteral as used herein includes, subcutaneous, intravenous,intramuscular, intrasternal infusion techniques, or intraperitoneally.Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable non-irritating excipient such as cocoabutter and polyethylene glycols that are solid at ordinary temperaturesbut liquid at the rectal temperature and will therefore melt in therectum and release the drug.

The dosage regimen for treating a disorder or a disease with the vectorsof this invention and/or compositions of this invention is based on avariety of factors, including the type of disease, the age, weight, sex,medical condition of the patient, the severity of the condition, theroute of administration, and the particular compound employed. Thus, thedosage regimen can vary widely, but can be determined routinely usingstandard methods.

The pharmaceutically active compounds of this invention can be processedin accordance with conventional methods of pharmacy to produce medicinalcompositions or agents for administration to patients, including humansand other mammals. For oral administration, the pharmaceuticalcomposition can be in the form of, for example, a liquid, an ocularinsert, a capsule, a tablet, a suspension. The pharmaceuticalcomposition is preferably made in the form of a dosage unit containing agiven amount of active agent. For example, these can contain an amountof vector from about 10³-10¹⁵ viral particles, preferably from about10⁶-10¹² viral particles. A suitable daily dose for a human or othermammal can vary widely depending on the condition of the patient andother factors, but, once again, can be determined using routine methods.Administration can be by injection of the active agent as a compositiontogether with suitable pharmacologically acceptable carriers such assaline, dextrose, or water.

A preferred method for inhibiting ocular neovascularization in a patientcomprises administering to a patient an ocular neovascularizationinhibiting amount of a water-soluble polypeptide of SEQ ID NO: 12, SEQID NO: 7, or an ocular neovascularization inhibiting fragment thereof,the fragment including at least one of amino acid residue signaturesequences HVGH (SEQ ID NO: 10) and KMSAS (SEQ ID NO: 11). Preferably,the administration is effected daily, weekly, monthly, quarterly orsemi-annually. When administration is effected daily, a daily dose ofabout 20 to about 100 micrograms per kilogram body weight of thepolypeptide is preferred. When administration is effected quarterly, aquarterly dose of about 2 to about 9 milligrams per kilogram body weightof the polypeptide is preferred. Preferably, the administration iseffected by intraocular delivery, such as intravitreal delivery. Theadministration can be effected by a sustained delivery device, by genetherapy, by cell-based ocular delivery, and the like.

While the nucleic acids and/or vectors of the invention can beadministered as the sole active pharmaceutical agent, they can also beused in combination with one or more vectors of the invention or otheragents. When administered as a combination, the therapeutic agents canbe formulated as separate compositions that are given at the same timeor different times, or the therapeutic agents can be given as a singlecomposition.

The polypeptide of the present invention can also be utilized incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the protein, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptide of the present invention can be employed on conjunction withother therapeutic compounds.

The pharmaceutical compositions can be administered in a convenientmanner, such as the intraocular, eye drop, and systemic routes. Theamounts and dosage regimens of the tRNA synthetase-derived polypeptidesadministered to a patient will depend on a number of factors, such asthe mode of administration, the nature of the condition being treated,the body weight of the subject being treated and the judgment of theprescribing physician. Generally speaking, the polypeptide isadministered in therapeutically effective doses of at least about 10μg/kg body weight. Preferably, the dosage is about 10 μg/kg body weightto about 1 mg/kg body weight daily, taking into the account thefrequency, routes of administration, symptoms, etc.

Angiostatic TrpRS therapy can be used to oppose the angiogenic activityof endogenous and exogenous angiogenic factors, and to prevent thefurther growth or even regress solid tumors, since angiogenesis andneovascularization are essential steps in solid tumor growth. Suchtherapies can also be used to treat rheumatoid arthritis, psoriasis anddiabetic retinopathy which are all characterized by abnormalangiogenesis.

Compositions are provided containing therapeutically effective amountsand concentrations of recombinant adenovirus delivery vectors fordelivery of therapeutic gene products to cells that express a particularreceptor. These cells include cells of the eye. Of particular interestare photoreceptor cells of the eye. Administration can be effected byany means through which contacting with the photoreceptors is effected.To provide access to photoreceptor cells, preferable modes ofadministration include, but are not limited to, subretinal injection orintravitreal injection.

The recombinant viral compositions can also be formulated forimplantation into the anterior or posterior chamber of the eye,preferably the vitreous cavity, in sustained released formulations, suchas adsorbed to biodegradable supports, including collagen sponges, or inliposomes. Sustained release formulations can be formulated for multipledosage administration, so that during a selected period of time, such asa month or up to about a year, several dosages are administered. Thus,for example, liposomes can be prepared such that a total of about two toup to about five or more times the single dosage is administered in oneinjection.

The vectors are formulated in an opthalmologically acceptable carrierfor intraocular, preferably intravitreal, administration in a volume ofbetween about 0.05 ml and 0.15 ml, preferably about 0.05 and 0.1 ml.

The compositions can be provided in a sealed sterile vial containing anamount of the active agent that upon intraocular administration deliversa sufficient amount of viral particles to the photoreceptors in a volumeof about 50 to 150 μl, containing at least about 10⁷, more preferably atleast about 10⁸ plaque forming units in such volume. Typically, thevials will, thus, contain about 0.15 ml of the composition.

To prepare such compositions, the viral particles are dialyzed into asuitable opthalmologically acceptable carrier or viral particles can beconcentration and/or mixed therewith. The resulting mixture can be asolution, suspension or emulsion. In addition, the viral particles canbe formulated as the sole pharmaceutically active ingredient in thecomposition or can be combined with other active agents for theparticular disorder treated.

For administration by intraocular injection or via eye drops, suitablecarriers include, but are not limited to, physiological saline,phosphate buffered saline (PBS), balanced salt solution (BSS), Ringerslactate solution, and solutions containing thickening and solubilizingagents, such as glucose, polyethylene glycol, and polypropylene glycoland mixtures thereof. Liposomal suspensions can also be suitable aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art. Suitable opthalmologicallyacceptable carriers are known. Solutions or mixtures intended forophthalmic use can be formulated as 0.01%-10% (w/w) isotonic solutions,pH about 5-7, with appropriate salts (see, e.g., U.S. Pat. No.5,116,868, which describes typical compositions of ophthalmic irrigationsolutions and solutions for local application). Such solutions, whichhave a pH adjusted to about 7.4, contain, for example, 90-100 mM sodiumchloride, 4-6 mM dibasic potassium phosphate, 4-6 mM dibasic sodiumphosphate, 8-12 mM sodium citrate, 0.5-1.5 mM magnesium chloride,1.5-2.5 mM calcium chloride, 15-25 mM sodium acetate, 10-20 mM sodiumD,L-β-hydroxybutyrate and 5-5.5 mM glucose.

The compositions can be prepared with carriers that protect the activeagent against rapid elimination from the body, such as time releaseformulations or coatings. Such carriers include controlled releaseformulations, such as, but not limited to, microencapsulated deliverysystems, and biodegradable, biocompatible polymers, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters,polylactic acid and other types of implants that can be placed directlyinto the anterior or posterior chamber or vitreous cavity of the eye.The compositions can also be administered in pellets, such as ELVAX®pellets (ethylene-vinyl acetate copolymer resin, DuPont).

Liposomal suspensions, including tissue-targeted liposomes, can also besuitable as pharmaceutically acceptable carriers. For example, liposomeformulations can be prepared by methods known to those of skill in theart (see, e.g., Kimm et al. Bioch. Bioph. Acta 728:339-398 (1983); Assilet al. Arch Opthalmol. 105:400 (1987); and U.S. Pat. No. 4,522,811). Theviral particles can be encapsulated into the aqueous phase of liposomesystems.

The active materials or agents can also be mixed with other activematerials, that do not impair the desired action, or with materials thatsupplement the desired action or have other action, includingviscoelastic materials, such as hyaluronic acid, which is sold under thetrademark HEALON® (Pharmacia, Inc), which is a solution of a highmolecular weight (MW) of about 3 millions fraction of sodium hyaluronate(see, e.g., U.S. Pat. Nos. 5,292,362, 5,282,851, 5,273,056, 5,229,127,4,517,295 and 4,328,803), and a resins sold under the trademark VISCOAT®(available from Alcon Surgical, Inc.), which are fluorine-containing(meth)acrylates, such as, 1H,1H,2H,2H-heptadecafluorodecylmethacrylate(see, e.g., U.S. Pat. Nos. 5,278,126, 5,273,751 and 5,214,080; andresins sold under the trademark ORCOLON® (Optical Radiation Corporation,see e.g., U.S. Pat. No. 5,273,056), and methylcelluloses, methylhyaluronates, polyacrylamides and polymethacrylamides (see e.g., U.S.Pat. No. 5,273,751). The viscoelastic materials are present generally inamounts ranging from about 0.5 to 5%, preferably 1 to 3% by weight ofthe conjugate material and serve to coat and protect the treatedtissues. The compositions can also include a dye, such as methylene blueor other inert dye, so that the composition can be seen when injectedinto the eye. Additional active agents can be included.

The compositions can be enclosed in ampules, disposable syringes ormultiple or single dose vials made of glass, plastic or other suitablematerial. Such enclosed compositions can be provided in kits. Inparticular, kits containing vials, ampules or other container,preferably disposable vials with sufficient amount of the composition todeliver about 0.1 ml thereof, and disposable needles, preferably selfsealing 25-33 gauge needles, or smaller, are provided herein.

Finally, the compositions can be packaged as articles of manufacturecontaining packaging material, typically a vial, an opthalmologicallyacceptable composition containing a polypeptide of the present inventionand a label that indicates the therapeutic use of the composition.

Also provided are kits for practice of the methods herein. The kitscontain one or more containers, such as sealed vials, with sufficientcomposition for single dosage administration, and one or more needles,such as self sealing 25-33 gauge or smaller needles, preferably 33 gaugeor smaller needles, with precisely calibrated syringes or otherprecisely calibrated delivery device, suitable for intravitrealinjection.

Administration of the composition is preferably by intraocularinjection, although other modes of administration can be effective, ifthe sufficient amount of the compound achieves contact with the vitreouscavity. Intraocular injection can be effected by intravitreal injection,aqueous humor injection or injection into the external layers of theeye, such as subconjunctival injection or subtenon injection, or bytopical application to the cornea, if a penetrating formulation is used.

For any particular patient, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of therecombinant viruses. The concentration and amount ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed methods.

The present invention also provides a method of assaying theangiogenesis inhibiting activity of a composition. The method comprisesinjecting a solution of a composition to be assayed into an eye of anewborn mouse on about day 7 or 8 postnatal (i.e., about 7 to 8 daysafter the mouse is born). The mouse is euthanized on about day 12 or 13postnatal and the injected eye is removed. The retina is excised fromthe injected eye and stained with a rabbit anti-mouse collagen IVantibody and a fluorescent-labeled goat anti-rabbit IgG antibody tovisualize the vascular network of the retina. The degree ofvascularization of the deep outer vascular plexus of the stained retinaexposed to the composition to be assayed is microscopically comparedwith the degree of vascularization of a retina from the eye of a controlmouse of the same age that has been similarly stained and which was notexposed to the composition. A substantially lower level ofvascularization in the retina of the mouse exposed to the composition tobe assayed relative to the control mouse indicates inhibition ofangiogenesis by that composition.

The Examples that follow are illustrative of specific embodiments of theinvention, and of various uses thereof. They are provided forillustrative and explanatory purposes only, but are not to be taken aslimiting.

EXAMPLE 1 Preparation of Endotoxin-Free Recombinant TrpRS

Endotoxin-free recombinant human TrpRS was prepared as follows. Plasmidsencoding full-length TrpRS (amino acid residues 1-471 of SEQ ID NO: 1),or truncated TrpRS, hereinafter referred to as T2 (SEQ ID NO: 12),consisting essentially of residues 94-471 of SEQ ID NO: 1 (i.e.,residues 94-471 of full-length TrpRS) and a second truncated TrpRS,hereinafter referred to as T1 (SEQ ID NO: 13), consisting essentially ofresidues 71-471 of SEQ ID NO: 1 were prepared. Each plasmid also encodeda C-terminal tag comprising six histidine residues (e.g. amino acidresidues 472-484 of SEQ ID NO: 1), and an initial methionine residue.The His₆-tagged T1 has the amino acid sequence of SEQ ID NO: 5, whereasthe His₆-tagged T2 has the amino acid sequence of SEQ ID NO: 7.

The above plasmids were introduced into E. coli strain BL 21 (DE 3)(Novagen, Madison, Wis.). Human mature EMAPII, also encoding aC-terminal tag of six histidine residues, was similarly prepared foruse. Overexpression of recombinant TrpRS was induced by treating thecells with isopropyl β-D-thiogalactopyranoside for 4 hours. Cells werethen lysed and the proteins from the supernatant purified on HIS●BIND®nickel affinity columns (Novagen) according to the manufacturer'ssuggested protocol. Following purification, TrpRS proteins wereincubated with phosphate-buffered saline (PBS) containing 1 μM ZnSO₄ andthen free Zn²⁺ was removed (Kisselev et al., Eur. J. Biochem. 120:511-17(1981)).

Endotoxin was removed from protein samples by phase separation usingTriton X-114 (Liu et al., Clin. Biochem. 30:455-63 (1997)). Proteinsamples were determined to contain less than 0.01 units of endotoxin permL using an E-TOXATE® gel-clot assay (Sigma, St. Louis, Mo.). Proteinconcentration was determined by the Bradford assay (Bio-Rad, Hercules,Calif.) using bovine serum albumin (BSA) as a standard.

EXAMPLE 2 Cleavage of Human TrpRS by PMN Elastase

Cleavage of human full-length TrpRS by PMN elastase was examined. TrpRSwas treated with PMN elastase in PBS (pH 7.4) at a protease:proteinratio of 1:3000 for 0, 15, 30, or 60 minutes. Following cleavage,samples were analyzed on 12.5% SDS-polyacrylamide gels. PMN elastasecleavage of a full-length TrpRS of about 53 kDa, encoded by nucleotides3428 to 4738 of DNA SEQ ID NO: 2) generated a major fragment of about 46kDa (SEQ ID NO: 5, T1 having the C-terminal histidine tag) and a minorfragment of about 43 kDa (SEQ ID NO: 7, T2 having the C-terminalhistidine tag).

Western blot analysis with antibodies directed against thecarboxyl-terminal His₆-tag of the recombinant TrpRS protein revealedthat both fragments possessed the His₆-tag at their carboxyl-terminus.Thus, only the amino-terminus of two TrpRS fragments has been truncated.The amino-terminal sequences of the TrpRS fragments were determined byEdman degradation using an ABI Model 494 sequencer. Sequencing of thesefragments showed that the amino-terminal sequences were S—N—H-G-P (SEQID NO: 8) and S-A-K-G-I (SEQ ID NO: 9), indicating that theamino-terminal residues of the major and minor TrpRS fragments werelocated at positions 71 and 94, respectively, of full-length TrpRS.These human TrpRS constructs are summarized in FIG. 1. Signaturesequences —HVGH— (SEQ ID NO: 10) and —KMSAS— (SEQ ID NO: 11) are shownin boxes.

The angiostatic activity of the major and minor TrpRS fragments wasanalyzed in angiogenesis assays. Recombinant forms of the major andminor TrpRS fragments SEQ ID NO: 5 and SEQ ID NO: 7 each having aC-terminal histidine tag (amino acid residues 472-484 of SEQ ID NO: 1)were used in these assays. Both TrpRS fragments were capable ofinhibiting angiogenesis.

EXAMPLE 3 Truncated Fragments of Trp-RS Show Potent Angiostatic Effectfor Retinal Angiogenesis

Angiostatic activity of truncated forms derived from tryptophanyl-rRNAsynthetase (TrpRS, 53 kDa; SEQ ID NO: 1) was examined, in a post-natalmouse retinal angiogenesis model Friedlander et al. Abstracts 709-B84and 714-B89, IOVS 41(4):138-139 (Mar. 15, 2000) has reported thatpostnatal retinal angiogenesis proceeds in stages in the mouse. Thepresent invention provides a method of assaying angiogenesis inhibitionby exploiting this staged retinal vascularization.

Endotoxin-free recombinant mini-TrpRS (48 kDa splice variant ofhistidine tagged TrpRS; SEQ ID NO: 3) and T2 (43 kDa cleavage product ofhistidine tagged TrpRS; SEQ ID NO: 7) were prepared as recombinantproteins. These proteins were injected intra-vitreally into neonatalBalb/C mice on postnatal (P) day 7 or 8 and the retinas harvested on P12or P13. Collagen IV antibody and fluorescein-conjugated secondaryantibody were used to visualize the vessels in retinal whole mountpreparations. Anti-angiogenic activity was evaluated by confocalmicroscopic examination based upon the effect of injected proteins onformation of the deep, outer, vascular plexus. Intra-vitreous injectionand retina isolation was performed with a dissecting microscope (SMZ645, Nikon, Japan). An eyelid fissure was created in postnatal day 7(P7) mice with a fine blade to expose the globe for injection of T2 (5pmol) or TrpRS (5 pmol). The samples (0.5 μl) were injected with asyringe fitted with a 32-gauge needle (Hamilton Company, Reno, Nev.).The injection was made between the equator and the corneal limbus;during injection the location of the needle tip was monitored by directvisualization to determine that it was in the vitreal cavity. Eyes withneedle-induced lens or retinal damage were excluded from the study.After the injection, the eyelids were repositioned to close the fissure.

On postnatal day 12 P12, animals were euthanized and eyes enucleated.After 10 minutes in 4% paraformaldehyde (PFA) the cornea, lens, sclera,and vitreous were excised through a limbal incision. The isolated retinawas prepared for staining by soaking in methanol for 10 minutes on ice,followed by blocking in 50% fetal bovine serum (Gibco, Grand Island,N.Y.) with 20% normal goat serum (The Jackson Laboratory, Bar Harbor,Me.) in PBS for 1 hour on ice. The blood vessels were specificallyvisualized by staining the retina with a rabbit anti-mouse collagen IVantibody (Chemicon, Temecula, Calif.) diluted 1:200 in blocking bufferfor 18 h at 4° C. An ALEXA FLUOR® 594-conjugated goat anti-rabbit IgGantibody (Molecular Probes, Eugene, Oreg.) (1:200 dilution in blockingbuffer) was incubated with the retina for 2 hours at 4° C. The retinaswere mounted with slow-fade mounting media M (Molecular Probes, Eugene,Oreg.).

Angiostatic activity was evaluated based upon the degree of angiogenesisin the deep, outer retinal vascular layer (secondary layer) that formsbetween P8 and P12. The appearance of the inner blood vessel network(primary layer) was evaluated for normal development and signs oftoxicity. None of the protein constructs used in this example producedany adverse effects on the primary layer.

FIG. 2 provides a photomicrographic depiction of the ability of T2 toinhibit vascularization of the secondary deep network of the mouseretina. In FIG. 2, row A shows the vascular network of a retina exposedto TrpRS, Row B shows the vascular network of a retina exposed toMini-TrpRS, and row C shows the vascular network of a retina exposed topolypeptide T2 of the present invention. The first (left) column showsthe primary superficial network, and the second column shows thesecondary deep network. As is evident from FIG. 2, none of thepolypeptides affected the primary superficial network, whereas only T2significantly inhibited vascularization of the secondary deep network.

Most PBS-treated eyes exhibited normal retinal vascular development, butcomplete inhibition of the outer vascular layer was observed in about8.2% (n=73) of the treated eyes. Complete inhibition of the outernetwork was observed in 28% of mini-TrpRS (0.5 mg/ml)-treated eyes(n=75). The smaller, truncated form (T2) was a far more potent inhibitorof angiogenesis in a dose dependent fashion; 14.3% were completelyinhibited after treatment with 0.1 mg/ml of T2 (n=14), 40% aftertreatment with 0.25 mg/ml (n=20) and 69.8% inhibited completely after0.5 mg/ml (n=53). The data for the 0.5 mg/ml treatments are presentedgraphically in FIG. 3. Extracts of mouse retina contain a protein withthe same apparent molecular mass and immunoreactivity as humanmini-TrpRS, as analyzed by SDS-PAGE and Western Blot. Full-length mouseand human TrpRS share about 88% amino acid identity and contain 475 and471 amino acids, respectively. Truncated forms of TrpRS, especially T2,have a potent angiostatic effect on retinal vascular development.

EXAMPLE 4 Matrigel Angiogenesis Assay

A mouse matrigel angiogenesis assay was used to examine the angiostaticactivity of T2 (SEQ ID NO: 7) according to the methods described byBrooks et al. Methods Mol. Biol., 129: 257-269 (1999) and Eliceiri etal. Mol. Cell, 4: 915-924 (1999). It was performed as described with thefollowing modifications. Athymic wehi mice were subcutaneously implantedwith 400 μl growth-factor depleted matrigel (Becton Dickinson, FranklinLakes, N.J.) containing 20 nM VEGF. The angiostatic activity of T2 wasinitially tested by including 2.5 μM T2 in the matrigel plug. Thepotency was determined by including various concentrations of T2 in theplug. On day 5, the mice were intravenously injected with thefluorescein-labeled endothelial binding lectin Griffonia (Bandeiraea)Simplicifolia I, isolectin B4 (Vector Laboratories, Burlingame, Calif.)and the matrigel plugs were resected. The fluorescein content of eachplug was quantified by spectrophotometric analysis after grinding theplug in RIPA buffer (10 mM sodium phosphate, pH 7.4, 150 mM sodiumchloride, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate).

EXAMPLE 5 Localization of T2 Binding within the Retina

To assess the uptake and localization of T2 injected into the retina,fluorescein-labeled (ALEXA® 488, Molecular Probes, Inc., Eugene Oreg.)T2 was injected into the vitreous of the eye on postnatal day 7 (P7).Globes were harvested on P8 and P12 and fixed in 4% PFA for 15 min. Theretinas were further dissected free of adherent nonretinal tissue andplaced in 4% PFA overnight at 4° C. and then embedded in medium(TISSUE-TEK® O.C.T., Sakura FineTechnical Co., Japan) on dry ice.Cryostat sections (10 micron) were rehydrated with PBS and blocked with5% BSA, 2% normal goat serum in PBS. Blood vessels were visualized withanti-mouse collagen IV antibody as described above. VECTASHIELD®containing DAPI nuclear stain (Vector Laboratories, Burlingame, Calif.)was used to mount the tissues with a cover slip.

Alternatively, unstained retina sections were incubated with 200 nMfluorescein-labeled full-length TrpRS or fluorescein-labeled T2 inblocking buffer overnight at 4° C. Sections were washed six times for 5minutes each in PBS, followed by incubation with 1 μg/ml DAPI for 5minutes for visualization of the nuclei. Pre-blocking with unlabeled T2was performed by incubating 1 μM unlabeled T2 for 8 hours at 4° C. priorto incubation with fluorescein-labeled T2. Retinas were examined with amultiphoton BioRad MRC 1024 confocal microscope. 3-D vascular imageswere produced from a set of Z-series images using the Confocal Assistantsoftware (BioRad, Hercules, Calif.).

Angiostatic Potency of T2 in the Mouse Matrigel Plug Assay. We examinedT2 (SEQ ID NO: 7) to determine whether it had angiostatic activity, eventhough it had lost aminoacylation activity. The mouse matrigel assay wasused to examine the angiostatic activity of T2 in vivo. VEGF₁₆₅-inducesthe development of blood vessels into the mouse matrigel plug. When T2was added to the matrigel along with VEGF₁₆₅, angiogenesis was blockedin a dose-dependent manner with a IC₅₀ of 1.7 nM as shown in FIG. 4.

Fluorescein-labeled T2 Localizes to Retinal Blood Vessels. In order tovisualize the intraocular localization of T2 (SEQ ID NO: 7), we examinedthe distribution of fluorescein-labeled T2 following intravitreousinjection on postnatal day 7. Retinas were isolated the following day,sectioned and examined using confocal microscopy. The distribution ofthe injected protein was restricted to blood vessels. This localizationwas confirmed by co-staining labeled T2 treated eyes with anfluorescein-labeled (ALEXA® 594) anti-collagen IV antibody (data notshown). Five days after injection of fluorescein-labeled T2 (on P12),the green fluorescence of the labeled T2 was still visible (FIG. 5A). Inthese retinas, no secondary vascular layer was observed at P12,indicating that the fluorescein-labeled T2 retained angiostatic activitycomparable to unlabeled T2. Retinas injected on P7 withfluorescein-labeled full-length TrpRS developed a secondary vascularlayer by P12 but no vascular staining was observed (FIG. 5B). In FIG. 5,fluorescein-labeled proteins are green, collagen-labeled vessels arered, and nuclei are blue.

To further evaluate the binding properties of labeled T2,cross-sectioned slices of normal neonatal retinas were stained withfluorescein-labeled T2. Under these conditions, fluorescein-labeled T2only bound to blood vessels (FIG. 5C). The binding was specific as itwas blocked by pre-incubation with unlabeled T2 (data not shown). Noretinal vessel staining was observed when fluorescein-labeledfull-length TrpRS was applied to the retinas (FIG. 5D), consistent withthe absence of angiostatic activity of the full-length enzyme.

As shown in FIG. 5, fluorescein-labeled T2 is angiostatic and localizesto retinal blood vessels. Fluorescein-labeled T2 (FIG. 5A) orfull-length TrpRS (FIG. 5B) were injected (0.5 μl, intravitreous) onpostnatal day 7 (P7). The retinas were harvested on P8 and stained withan anti-collagen IV antibody and DAPI nuclear stain, Labeled T2 (upperarrow pointing to vessel in FIG. 5A) localized to blood vessels in theprimary superficial network (1°). Note that the secondary deep networkis completely absent (2°). While both the primary (1°) and secondary(2°) vascular layers are present in eyes injected withfluorescein-labeled full-length TrpRS (arrows in FIG. 5B), no labelingis observed.

In a separate set of experiments, frozen sections of P15 retinas werestained with fluorescein-labeled T2 (FIG. 5C) or fluorescein-labeledfull-length TrpRS (FIG. 5D) and imaged in the confocal scanning lasermicroscope. Labeled T2 selectively localized to blood vessels andappears as a bright green vessel penetrating the primary and secondaryretinal vascular layers just below the label “2°” in FIG. 5C. Nostaining was observed with full-length TrpRS (FIG. 5D).

Full-length TrpRS contains a unique NH₂-terminal domain and lacksangiostatic activity. Removing part or all of this entire domain revealsa protein with angiostatic activity. The structures responsible forangiostatic activity of T2 appear to be contained within the coreRossmann fold nucleotide binding domain. The NH₂-terminal domain, whichcan be deleted by alternative splicing or by proteolysis, may regulatethe angiostatic activity of TrpRS, possibly by revealing a binding sitenecessary for angiostasis that is inaccessible in full-length TrpRS.

VEGF-induced angiogenesis in the mouse matrigel model was completelyinhibited by T2 as was physiological angiogenesis in the neonatalretina. Interestingly, the most potent anti-angiogenic effect of TrpRSfragments in vitro and in CAM and matrigel models is observed inVEGF-stimulated angiogenesis. The neonatal mouse retinal angiogenesisresults are consistent with a link between VEGF-stimulated angiogenesisand the angiostatic effects of TrpRS fragments; retinal angiogenesis inthis system may be driven by VEGF. In addition, the inhibition observedin the retinal model was specific for newly developing vessels;pre-existing (at the time of injection) primary vascular layer vesselswere unaltered by the treatment. While the mechanism for the angiostaticactivity of T2 is not known, the specific localization of T2 to theretinal endothelial vasculature and the selective effect of T2 on newlydeveloping blood vessels suggest that T2 may function through anendothelial cell receptor expressed on proliferating or migrating cells.Further understanding of the mechanism of T2 angiostatic activityrequires identification of the relevant cell receptor.

A variety of cell types that produce, upon interferon-γ stimulation, theangiostatic mini TrpRS also produce angiostatic factors such as IP-10.Thus, these results raise the possibility of a role for TrpRS in normal,physiologically relevant pathways of angiogenesis. Another ubiquitouscellular protein, pro-EMAPII (p43), has two apparently unrelated rolessimilar to those reported here for TrpRS. Pro-EMAPII assists proteintranslation by associating with the multisynthetase complex of mammalianaminoacyl tRNA synthetases. It is processed and secreted as EMAPII, anda role for EMAPII as an angiostatic mediator during lung development hasbeen suggested.

Thus, T2 can be utilized in physiologically relevant angiogenicremodeling observed under normal or pathological conditions. In normalangiogenesis, T2 can aid in establishing physiologically importantavascular zones present in some organs such as the foveal avascular zoneof the central retina. Pathological angiogenesis can occur if thecleavage of full-length TrpRS was inhibited, leading to an overgrowth ofvessels.

In ocular diseases, neovascularization can lead to catastrophic loss ofvision. These patients can potentially receive great benefit fromtherapeutic inhibition of angiogenesis. Vascular endothelial growthfactor has been associated with neovascularization and macular edema inthe retina, although it is believed that other angiogenic stimuli alsohave roles in retinal angiogenesis. We have observed an associationbetween VEGF-stimulated angiogenesis and potent angiostatic activity ofTrpRS fragments, making these molecules useful in the treatment ofhypoxic, and other, proliferative retinopathies. There has been noreport in the literature of an anti-angiogenic agent that completelyinhibits angiogenesis 70% of the time, as does the T2 of the presentinvention (FIG. 5). Another advantage of TrpRS fragments is that theyrepresent naturally occurring and, therefore, potentiallynon-immunogenic, anti-angiogenics. Thus, these molecules can bedelivered via targeted cell- or viral vector-based therapy. Because manypatients with neovascular eye diseases have associated systemic ischemicdisease, local anti-angiogenic treatment with genetically engineeredcells or viral vectors placed directly into the eye is desirable.

In addition to treatment of angiogenic retinopathies, the TrpRSfragments of the present invention, particularly T2 and angiogenesisinhibiting fragments thereof, can also inhibit solid tumor growth bypreventing vascularization of the tumor. The TrpRS fragments of thepresent invention block VEGF-induced proliferation and chemotaxis ofendothelial cells in vitro, and are thus useful in the treatment of anypathology involving unwanted endothelial cell proliferation andvascularization.

1. A method for inhibiting ocular neovascularization in a patient, themethod comprising administering to a patient an ocularneovascularization inhibiting amount of a water-soluble polypeptideselected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 7, andan ocular neovascularization inhibiting fragment thereof, the fragmentincluding at least one of amino acid residue signature sequences HVGH(SEQ ID NO:10) and KMSAS (SEQ ID NO:11).
 2. The method of claim 1wherein the administration is effected daily.
 3. The method of claim 1wherein the administration is effected weekly.
 4. The method of claim 1wherein the administration is effected monthly.
 5. The method of claim 1wherein the administration is effected quarterly.
 6. The method of claim1 wherein the administration is effected semi-annually.
 7. The method ofclaim 1 wherein a daily dose of about 20 to about 100 micrograms perkilogram body weight of the polypeptide is administered to the patient.8. The method of claim 1 wherein a quarterly dose of about 2 to about 9milligrams per kilogram body weight of the polypeptide is administeredto the patient.
 9. The method of claim 1 wherein the administration iseffected by intravitreal delivery.
 10. The method of claim 1 wherein theadministration is effected by intraocular delivery.
 11. The method ofclaim 1 wherein the administration is effected by a sustained deliverydevice.
 12. The method of claim 1 wherein the administration is effectedby gene therapy.
 13. The method of claim 1 wherein the administration iseffected by cell-based ocular delivery.
 14. The method of claim 1wherein the water-soluble polypeptide consists of SEQ ID NO:
 12. 15. Amethod for inhibiting ocular neovascularization in a patient, the methodcomprising intraocularly injecting into the eye of a patient an ocularneovascularization inhibiting amount of a water-soluble polypeptideconsisting of SEQ ID NO: 12, or an ocular neovascularization inhibitingfragment thereof, the fragment including at least one of amino acidresidue signature sequences HVGH (SEQ ID NO:10) and KMSAS (SEQ IDNO:11).
 16. The method of claim 15 wherein the water-soluble polypeptideconsists of SEQ ID NO:
 12. 17. The method of claim 15 wherein thewater-soluble polypeptide consists of a fragment of SEQ ID NO: 12comprising the signature sequence SEQ ID NO: 10 at the N-terminusthereof and SEQ ID NO:11 at the C-terminus thereof.